autologous blood and marrow transplantation - Blog Science ...

Bewick et al. 365
Blood samples obtained during the course of treatment were collected in
ethylene-diamine tetraacetic acid (EDTA) vacutainer tubes. Plasma was collected
by centrifugation at 400g for 10 minutes. The plasma was removed, frozen in
aliquots, and stored at -70°C until required for analysis. Plasma HER-2 levels were
examined at three different time points: 1) at initial enrollment to the clinical
program; 2) at the time of first or second apheresis (following ICT with rhGM-CSF
or rhG-CSF); and 3) ~1 month after HDCT and ABSC reinfusion. Before assay,
frozen plasma samples were thawed gradually and centrifuged at 8000 rpm for 5
minutes in Eppendorf tubes. Protein concentration was measured by the Bradford
protein dye method, 28
using bovine serum albumin as the standard.
Measurements of HER-2 levels in plasma of seven healthy women resulted in a
mean concentration of 13.8 U/mL ± 2.3 SD. To further offset any variation due to
patient plasma differences during treatment, data was expressed as U/mg plasma
protein or 0.134 U/mg protein ± 0.02. The cut-point for positivity (i.e., overexpression)
was defined as 21 U/mL or 0.2 U/mg protein (using the mean plus three
times the standard deviation calculation from Isola et al. 24
). Plasma samples
expressing HER-2 levels below this cut-point were defined as "HER-2 negative"
and those samples equal to or above this cut-point were defined as "HER-2
positive." More significant differences between the HER-2 positive and negative
patient populations were found when the data were calculated and analyzed using
the 0.2 U/mg protein cut-point and all data was subsequently evaluated using this
cut-point.
PATIENT EVALUATION AND STATISTICAL ANALYSIS
Progression free survival was defined as the time from study entry to
documented signs of recurrence or progression of disease. Overall survival was
defined as the time from study entry to death due to MBC. Survival curves were
calculated using the Kaplan-Meier method. Differences between survival times
were analyzed by the log-rank, Wilcoxon, and Cox tests for survivorship functions
and hazard ratios were calculated by Cox's proportional hazards method for
univariate analysis.
All patients had a physical and radiological evaluation based on World Health
Organization criteria at various times during the course of treatment and
subsequently every 2-3 months for 2 years and then every 6 months until evidence
of relapse.
Patients were considered to have achieved a complete response (CR) if no
evidence of disease was found for at least 4 weeks. Partial response (PR) was
defined as a reduction in measurable disease volume to